Fucosyllactose as breast milk identical non-digestible oligosaccharide with new functional benefit

ABSTRACT

The invention concerns nutritional compositions with fucosyllactose for use in stimulation of NK cells. The composition is suitable for infants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.13/383,822, filed Mar. 28, 2012, which is the U.S. National StageApplication of PCT/NL2010/50447, filed Jul. 12, 2010, which claims thebenefit of U.S. Provisional Patent Application No. 61/256,453, filedOct. 30, 2009, and European Patent Application No. 09165485.5, filedJul. 15, 2009, all of which are incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to infant nutrition with non-digestibleoligosaccharides, in particular to the use thereof for stimulating theimmune system.

BACKGROUND OF THE INVENTION

Human milk fed infants have a lower incidence of infections, includingviral infections, than formula fed infants. Many components in humanmilk, including immunoglobulins (such as IgA), interleukin (IL)-1, IL-6,IL-8, IL-10, interferon-γ (IFN-γ), immunocompetent cells, transforminggrowth factor-β (TGF-β), lactoferrin, nucleotides, and non-digestibleoligosaccharides (NDO) are thought to be involved in protection againstinfection with enteric or respiratory pathogens. TGF-β and dietarynucleotides were found to be components which may be responsible forincrease in natural killer cell activity.

NDO are a major constituent of human milk and are a major element of theinnate immune system of human milk. Human NDO promote the growth of abeneficial microbiota dominated by bifidobacteria and lactobacilli. Somehuman NDO are also known to be able to prevent directly the adhesion ofpathogens and toxins.

Human milk is the preferred food for infants. However, it is not alwayspossible or desirable to breast feed an infant. In such cases infantformulae or follow on formulae are a good alternative.

These formulae should have an optimal composition in order to mimic thebeneficial effects of breast milk as close as possible.

WO 2007/067053 discloses infant formula comprising the plant-derivedprebiotics inulin and galacturonic acid oligosaccharide and the fromlactose synthesized prebiotic transgalacto-oligosaccharide to reduceinfections.

WO 2007/010084 discloses mannan-poly- and oligosaccharides for immunestimulation.

U.S. Pat. No. 6,576,251 discloses a carbohydrate mixture for dieteticfoods administered by the enteral or parenteral route consisting of (a)monosaccharide(s), (b) oligosaccharide(s) (at most hexasaccharides) and(c) polysaccharide(s) (at least heptasaccharides), where the mixingratio a, b, c, in respect of weight, is: alpha=1, b=40 to 1000, and c=1to 50, and containing at least 1 weight percent of fucose occurringeither freely and/or bound to an oligosaccharide and/or apolysaccharide. The carbohydrate mixture is said to have both anutritional and a biological effect which is considerably greater thanthe corresponding action of the individual constituents.

EP 1 629 850 provides a method and composition for the treatment and/orprevention of respiratory tract infection and/or respiratory tractinfection disease, said method comprising orally administering acomposition to a mammal, said composition comprising a galactosecontaining indigestible oligosaccharide and at least 5 wt. % digestiblegalactose saccharide.

EP 2 072 052 relates to a composition suitable for use in the preventionof opportunistic infections in immune-compromised individuals comprisinga probiotic and a fucosylated oligosaccharide selected from the groupcomprising 2′-fucosyllactose, 3′fucosyllactose, difucosyllactose,lacto-N-fucopentaose, lacto-N-fucohexaose, fucosyllacto-N-hexaose andfucosyllacto-N-neohexaose. The document further discloses the use ofsuch a composition in the prevention of opportunistic infections inimmune-compromised individuals.

SUMMARY OF THE INVENTION

Human milk differs from milk from domestic animals in that it comprisesmore NDO and in that the NDO are structurally different. The group ofhuman NDO is very complex, since it represents a heterogenic group ofmore than 130 different compounds with diverse sugar composition.Because of their complex and polymorphic structure, large-scalesynthesis is complicated. It is therefore not yet technically andeconomically feasible to prepare infant nutrition with an NDOcomposition identical to human milk.

Recently, new techniques have become available to chemically synthesisespecific types of NDO identical to specific human NDO, thereby offeringthe opportunity to test the immunomodulatory capacity of specific humanNDO in in vitro and in vivo assays.

The inventors unexpectedly have found that fucosyllactose (FL), anoligosaccharide abundantly present in human breast milk and with arelatively simple structure, specifically increases the number andthereby the activity of Natural Killer (NK) cells. NK cells play animportant role in the natural defence against viral infections andtumour cells. The finding which specific oligosaccharide is responsiblefor increasing NK cell activity now enables the design of nutritionalcompositions comprising FL, more particularly 2′-FL, for the use ofincreasing NK cells and/or NK cell activity.

DETAILED DESCRIPTION

The present invention thus concerns a method for stimulating NK cellactivity and/or NK cell proliferation in a subject, said methodcomprising administering a composition comprising fucosyllactose to saidsubject, said composition not being human milk. In one embodiment thepresent method is a non-medical method for stimulating NK cell activityand/or NK cell proliferation in a subject. In one embodiment the presentmethod is for treating and/or preventing viral infections in a subject.

The present invention also concerns a method for treating and/orpreventing viral infections in a subject, said method comprisingadministering a composition comprising fucosyllactose to said subject,said composition not being human milk.

The invention can also be worded as the use of fucosyllactose in themanufacture of a composition for stimulating NK cell activity and/or NKcell proliferation, said composition not being human milk. In oneembodiment the composition is for treating and/or preventing viralinfections.

The invention can also be worded as the use of fucosyllactose in themanufacture of a composition for treating and/or preventing viralinfections, said composition not being human milk.

The invention can also be worded as a composition comprisingfucosyllactose for stimulating, in particular for use in stimulating, NKcell activity and/or NK cell proliferation, said composition not beinghuman milk. In one embodiment the composition is for use in treatingand/or preventing, viral infections.

The invention can also be worded as a composition comprisingfucosyllactose for treating and/or preventing, in particular for use intreating and/or preventing, viral infections, said composition not beinghuman milk.

The invention also concerns a method for treating and/or preventinginfections, by stimulating natural killer (NK) cell activity and/or NKcell proliferation, said method comprising administering a compositioncomprising fucosyllactose to said subject, said composition not beinghuman milk.

The invention can also be worded as the use of fucosyllactose in themanufacture of an enteral composition for treating and/or preventinginfections, by stimulating natural killer (NK) cell activity and/or NKcell proliferation, said composition not being human milk.

The invention can also be worded as a composition comprisingfucosyllactose for treating and/or preventing infections, in particularfor use in and/or preventing infections, by stimulating natural killer(NK) cell activity and/or NK cell proliferation, said composition notbeing human milk.

The invention also concerns a method for enhancing vaccination response,said method comprising administering a composition comprisingfucosyllactose to said subject, said composition not being human milk.In one embodiment the method is for enhancing vaccination response tovaccination with viral antigens.

The invention can also be worded as the use of fucosyllactose in themanufacture of an enteral composition for enhancing vaccinationresponse. In one embodiment the composition is for enhancing vaccinationresponse to vaccination with viral antigens.

The invention can also be worded as a composition comprisingfucosyllactose for enhancing, in particular for use in enhancing,vaccination response. In one embodiment the composition is for enhancingvaccination response to vaccination with viral antigens.

The composition that is administered according to the present method, orthat is used according to the present invention, is preferably enterallyadministered, more preferably orally. Or in other words the compositionis preferably for enteral, preferably oral administration or in otherwords the composition is an enteral, preferably oral, composition.

Fucosyllactose

The present composition comprises fucosyllactose. Fucosyllactose (FL) isa non-digestible oligosaccharide present in human milk. It is notpresent in bovine milk. It consists of three monose units, fucose,galactose and glucose linked together. Galactose linked to glucose via abeta 1→4 linkage is called lactose. A fucose unit is linked to agalactose unit of a lactose via an alpha 1,2 linkage (2′-fucosyllactose,2′-FL) or to the glucose unit of lactose via an alpha 1,3 linkage(3-fucosyllactose, 3-FL). The present composition preferably comprises2′-FL.

2′-FL, preferably α-L-Fuc-(1→2)-β-D-Gal-(1→4)-D-Glc, and 3-FL,preferably α-L-Fuc-(1→3)-[β-D-Gal-(1→4)]-D-Glc), are commerciallyavailable for instance from Sigma-Aldrich. Alternatively, they can beisolated from human milk, for example as described in Andersson &Donald, 1981, J Chromatogr. 211:170-1744, or produced by geneticallymodified micro-organisms, for example as described in Albermann et al,2001, Carbohydrate Res. 334:97-103.

Preferably, the composition comprises 1 mg to 3 g fucosyllactose per 100ml, more preferably 10 mg to 2 g, even more preferably 20 mg to 100 mgFL per 100 ml. Based on dry weight, the composition preferably comprises0.007 wt % to 20 wt % fucosyllactose, more preferably 0.07 wt % to 10 wt%, even more preferably 0.15 wt % to 1 wt %. A lower amount offucosyllactose will be less effective in increasing NK cells and/orincreasing NK cell activity, whereas a too high amount will result inunnecessary high costs of the product.

Non-Digestible Oligosaccharides Other than FL

The present composition preferably comprises non-digestibleoligosaccharides (NDO) other than FL. Preferably the NDO other than FLstimulate the growth of bifidobacteria and/or lactobacilli, morepreferably bifidobacteria. An increased content of bifidobacteria and/orlactobacilli stimulate the formation of a healthy intestinal microbiota.The NDO are preferably not or only partially digested in the intestineby the action of acids or digestive enzymes present in the human upperdigestive tract, in particular in the small intestine and stomach, andare fermented by the human intestinal microbiota. For example, sucrose,lactose, maltose and the common maltodextrins are considered digestible.

Preferably the present composition comprises non-digestibleoligosaccharides with a DP in the range of 2 to 250, more preferably 2to 60. The non-digestible oligosaccharide is preferably at least one,more preferably at least two, preferably at least three selected fromthe group consisting of fructo-oligosaccharides,galacto-oligosaccharides, xylo-oligosaccharides,arabino-oligosaccharides, arabinogalacto-oligosaccharides,gluco-oligosaccharides, chito-oligosaccharides,glucomanno-oligosaccharides, galactomanno-oligosaccharides,mannan-oligosaccharides, sialic acid comprising oligosaccharides, anduronic acid oligosaccharides. The group of fructo-oligosaccharidesincludes inulins, the group of galacto-oligosaccharides includestransgalacto-oligosaccharides or beta-galacto-oligosaccharides, thegroup of gluco-oligosaccharides includes cyclodextrins, gentio- andnigero-oligosaccharides and non-digestible polydextrose, the group ofgalactomanno-oligosaccharides includes partially hydrolyzed guar gum,and the group of uronic acid oligosaccharides includes galacturonic acidoligosaccharides and pectin degradation products.

More preferably the present composition comprises at least one, morepreferably at least two, most preferably three selected from the groupconsisting of fructo-oligosaccharides, beta-galacto-oligosaccharides anduronic acid oligosaccharides. More preferably the composition comprisesbeta-galacto-oligosaccharides.

In a preferred embodiment the composition comprises a mixture of inulinand short chain fructo-oligosaccharides. In a preferred embodiment thecomposition comprises a mixture of galacto-oligosaccharides andfructo-oligosaccharides selected from the group consisting of shortchain fructo-oligosaccharides and inulin, more preferably inulin. Amixture of at least two different non-digestible oligosaccharidesadvantageously stimulates the beneficial bacteria of the intestinalmicrobiota to a greater extent. Preferably the weight ratio in a mixtureof the two different non-digestible oligosaccharides, preferablygalacto-oligosaccharides and fructo-oligosaccharide, is between 25 and0.05, more preferably between 20 and 1. Galacto-oligosaccharides,preferably beta-galacto-oligosaccharides, are more capable ofstimulating bifidobacteria. Preferably the present composition comprisesgalacto-oligosaccharides, preferably beta-galacto-oligosaccharides, witha degree of polymerization (DP) of 2 to 10 and/orfructo-oligosaccharides with a DP of 2 to 60.

The galacto-oligosaccharides preferably arebeta-galacto-oligosaccharides. In a particularly preferred embodimentthe present composition comprises beta-galacto-oligosaccharides([galactose]n-glucose; wherein n is an integer ranging from 2 to 60,i.e. 2, 3, 4, 5, 6, . . . , 59 ,60; preferably n is selected from 2, 3,4, 5, 6, 7, 8, 9, and 10), wherein the galactose units are in majoritylinked together via a beta linkage. Beta-galacto-oligosaccharides arealso referred to as trans-galacto-oligosaccharides (TOS).Beta-galacto-oligosaccharides are for example sold under the trademarkVivinal™ (Borculo Domo Ingredients, Netherlands). Another suitablesource is Bi2Munno (Classado). Preferably the TOS comprises at least 80%beta-1,4 and beta-1,6 linkages based on total linkages, more preferablyat least 90%.

Fructo-oligosaccharide is a NDO comprising a chain of beta-linkedfructose units with a DP or average DP of 2 to 250, more preferably 2 to100, even more preferably 10 to 60. Fructo-oligosaccharide includesinulin, levan and/or a mixed type of polyfructan. An especiallypreferred fructo-oligosaccharide is inulin. Fructo-oligosaccharidesuitable for use in the compositions is also commercially available,e.g. Raftiline®HP (Orafti). Preferably the fructo-oligosaccharide has anaverage DP above 20.

Uronic acid oligosaccharides are preferably obtained from pectindegradation products. Hence the present composition preferably comprisesa pectin degradation product with a DP of 2 to100. Preferably the pectindegradation product is prepared from apple pectin, beet pectin and/orcitrus pectin. Preferably the uronic acid oligosaccharide is agalacturonic acid oligosaccharide. Preferably the composition comprisesFL and one of the group selected from galacto-oligosaccharide and uronicacid oligosaccharide.

Besides FL, most preferably the composition comprisesbeta-galacto-oligosaccharide, fructo-oligosaccharide and a uronic acidoligosaccharide. It was found that such a combination actssynergistically with fucosyllactose, in particular 2′-fucosyllactose.The weight ratiobeta-galacto-oligosaccharide:fructo-oligosaccharide:uronic acidoligosaccharide is preferably (20 to 2):1:(1 to 20), more preferably (20to 2):1:(1 to 10), even more preferably (20 to 2):1:(1 to 3), even morepreferably (12 to 7):1:(1 to 2). Most preferably the weight ratio isabout 9:1:1.1. Preferably the weight ratio FL tobeta-galacto-oligosaccharide, preferably TOS, is from 5 to 0.05, morepreferably 5 to 0.1, more preferably from 2 to 0.1. Preferably theweight ratio FL to fructo-oligosaccharide, preferably inulin, is from 10to 0. 05, more preferably 10 to 0.1, more preferably from 2 to 0.5.Preferably the weight ratio FL to uronic acid oligosaccharide,preferably derived from pectin, is from 10 to 0. 05, more preferably 10to 0.1 more preferably from 2 to 0.5.

Preferably, the composition comprises 80 mg to 4 g non-digestibleoligosaccharides, including fucosyllactose, per 100 ml, more preferably150 mg to 2 g, even more preferably 300 mg to 1 g non-digestibleoligosaccharides per 100 ml. Based on dry weight, the compositionpreferably comprises 0.25 wt % to 25 wt % non-digestibleoligosaccharides including fucosyllactose, more preferably 0.5 wt % to10 wt %, even more preferably 1.5 wt % to 7.5 wt %. A lower amount ofnon-digestible oligosaccharides will be less effective in stimulatingthe beneficial bacteria in the microbiota, whereas a too high amountwill result in side-effects of bloating and abdominal discomfort.

Nutritional Composition

Preferably the composition comprising fucosyllactose is a nutritionalcomposition. The composition of the present invention is not human milk.The present composition is preferably enterally administered, morepreferably orally.

The present composition is preferably a nutritional formula, preferablyan infant formula. The present composition can be advantageously appliedas a complete nutrition for infants. The present composition preferablycomprises a lipid component, protein component and carbohydratecomponent and is preferably administered in liquid form. The presentinvention includes dry food, preferably a powder, which is accompaniedwith instructions as to admix said dry food mixture with a suitableliquid, preferably with water.

The present invention advantageously provides a composition wherein thelipid component provides 5 to 50% of the total calories, the proteincomponent provides 5 to 50% of the total calories, and the digestiblecarbohydrate component provides 15 to 85% of the total calories. Thepresent invention advantageously provides a composition wherein thelipid component provides 20 to 50% of the total calories, the proteincomponent provides 5 to 30% of the total calories, and the digestiblecarbohydrate component provides 30 to 70% of the total calories.Preferably, in the present composition the lipid component provides 35to 50% of the total calories, the protein component provides 7.5 to12.5% of the total calories, and the digestible carbohydrate componentprovides 40 to 55% of the total calories. For calculation of the % oftotal calories for the protein component, the total of energy providedby the proteins, peptides and amino acids needs to be taken intoaccount.

The present composition preferably comprises at least one lipid selectedfrom the group consisting of animal lipid, excluding human lipids, andvegetable lipids. Preferably the present composition comprises acombination of vegetable lipids and at least one oil selected from thegroup consisting of fish oil, animal oil, algae oil, fungal oil, andbacterial oil. The present composition preferably comprises long chainpoly-unsaturated fatty acids (LC-PUFA). LC-PUFA are fatty acids or fattyacyl chains with a length of 20 to 24 carbon atoms, preferably 20 or 22carbon atoms comprising two or more unsaturated bonds. More preferablythe present composition comprises eicosapentaenoic acid (EPA, n-3),docosahexaenoic acid (DHA, n-3) and/or arachidonic acid (ARA, n-6).

Preferably the present composition comprises at least 0.1 wt. %,preferably at least 0.25 wt. %, more preferably at least 0.6 wt. %, evenmore preferably at least 0.75 wt. % LC-PUFA with 20 and 22 carbon atomsbased on total fat content.

The content of LC-PUFA, particularly the LC-PUFA with 20 and 22 carbonatoms, preferably does not exceed 6 wt %, more preferably does notexceed 3 wt. % of the total fat content as it is desirable to mimichuman milk as closely as possible. The LC-PUFA may be provided as freefatty acids, in triglyceride form, in diglyceride form, in monoglycerideform, in phospholipid form, or as a mixture of one of more of the above.The present composition preferably comprises between 5 and 75 wt. %polyunsaturated fatty acids based on total fat, preferably between 10and 50 wt. %.

The protein used in the nutritional composition is preferably selectedfrom the group consisting of non-human animal proteins (preferably milkproteins), vegetable proteins (preferably soy protein and/or riceprotein), hydrolysates thereof, free amino acids and mixtures thereof.The present composition preferably contains casein, whey, hydrolyzedcasein and/or hydrolyzed whey protein. Preferably the protein comprisesintact proteins, more preferably intact bovine whey proteins and/orintact bovine casein proteins.

The present composition preferably contains digestible carbohydratesselected from the group consisting of sucrose, lactose, glucose,fructose, corn syrup solids, starch and maltodextrins, more preferablylactose.

In view of the above, it is also important that the liquid food does nothave an excessive caloric density, however still provides sufficientcalories to feed the subject. Hence, the liquid food preferably has acaloric density between 0.1 and 2.5 kcal/ml, even more preferably acaloric density of between 0.5 and 1.5 kcal/ml, most preferably between0.6 and 0.8 kcal/ml.

Preferably the present composition comprises nucleotides and/ornucleosides, more preferably nucleotides. Preferably, the compositioncomprises cytidine 5′-monophospate, uridine 5′-monophospate, adenosine5′-monophospate, guanosine 5′-monophospate, and/or inosine5′-monophospate, more preferably cytidine 5′-monophospate, uridine5′-monophospate, adenosine 5′-monophospate, guanosine 5′-monophospate,and inosine 5′-monophospate. Preferably the composition comprises 5 to100, more preferably 5 to 50 mg, most preferably 10 to 50 mg nucleotidesand/or nucleosides per 100 gram dry weight of the composition. Thepresence of nucleotides and/or nucleotides advantageously stimulates NKcell activity. The nucleotides and/or nucleosides are deemed to actsynergistically with the fucosyllactose of the present composition.

Application

In one embodiment the present composition is used for stimulatingnatural killer cell activity and/or natural killer cell proliferation.In one embodiment the present composition is used for treating and/orpreventing viral infections. In one embodiment the composition forstimulating natural killer cell activity and/or natural killer cellproliferation, and/or for treating and/or preventing viral infections isfor administering to HIV patients, elderly and/or oncology patients.

NK cells are a type of cytotoxic lymphocytes that constitute a majorcomponent of the innate immune system. NK cells play a major role indefense against intracellular infections. NK-cells are defined as largegranular lymphocytes that do not express T-cell antigen receptors (TCR)or Pan T marker CD3 or surface immunoglobulins (Ig) B cell receptor butthat usually express the surface markers CD16 (FcγRIII) and CD56 inhumans. They were named “natural killers” because of the initial notionthat they do not require activation in order to kill cells that aremissing “self” markers of major histocompatibility complex (MHC) classI. NK cells have two major types of effector function; cell killing andthe secretion of cytokines Increasing NK cell activity (by increasingthe number of NK cells and/or by increasing the specific activity of anNK cell), results in an increased resistance against viral infections.The use of a nutritional composition comprising fucosyllactose istherefore preferably for preventing and/or treating viral infections,more preferably viral infections caused by orthomyxoviridae, inparticular the influenza virus, herpesviridae, rotavirus,cytomegalovirus, caliciviridae, respiratory syncytial virus, humanimunodeficiency virus and/or rhinovirus. The use of a nutritionalcomposition comprising fucosyllactose is therefore preferably forpreventing and/or treating viral infections, more preferably the viralinfections common cold, flu, measles, chicken pox, viral diarrhoea,viral gastro-enteritis, HIV infection and/or viral respiratory tractinfections. In a preferred embodiment the present invention is used forHIV patients. In one embodiment, the present invention concernsproviding nutrition to a HIV patient. The present composition isadvantageous for HIV patients since HIV patients have a decreasednatural killer cell activity.

The use of a nutritional composition comprising fucosyllactose istherefore especially beneficial for infant formula. In one embodiment,the present invention concerns providing nutrition to an infant. Formulafed infants have an underdeveloped immune system compared with adultsand are more prone to viral infections than human milk fed infants.Preferably the infant is from 0 to 36 months of age, more preferably of0 to 18 months, even more preferably of 0 to 12 months, most preferablyof 0 to 6 months of age. The younger the infant is, the less developedthe immune system.

The composition comprising fucosyllactose even more advantageously isused in preterm infants and/or very low or low birth weight infants,since these infants are even more vulnerable and/or prone to viralinfections.

The composition comprising fucosyllactose even more advantageously isused in infants delivered via Caesarean section. Caesarean section borninfants are born in a hospital in an environment having more pathogensagainst which the antibodies, conferred by the mother to the infant, arenot effective against. Caesarean section born infants have a delayed andless optimal colonization of the large intestinal tract and thereforeare also more prone to intestinal infections.

The composition comprising fucosyllactose is advantageously used fornutrition for elderly. In one embodiment, the present invention concernsproviding nutrition to an elderly person. An elderly person is a personhaving an age of 55 years or more, in particular of the age of 65 ormore. Elderly have a demonstrated lower activity of natural killer cellactivity than healthy young adult individuals. Elderly are especiallyvulnerable to viral infection complications. In a preferred embodimentthe present invention is used for treatment and/or prevention ofimmunosenescence in elderly. Elderly are more prone to the developmentof tumours. NK cell activity suppresses tumour cell proliferation. In apreferred embodiment the present invention is used for nutrition incancer patients. Oncology patients have a lower natural killer cellactivity than healthy young adult individuals.

EXAMPLES Example 1

Materials and Methods

6-8 Weeks old female C57BL/6 mice (Charles River) received semi-purifiedAIN-93G-based diets (Research Diet Service, Wijk bij Duurstede, theNetherlands), comprising

1) 2 wt % beta-galacto-oligosaccharide (GOS; source Vivinal GOS, BorculoDomo), fructo-oligosaccharide (FOS; source RaftilineHP, Orafti) andgalacturonic acid oligosaccharide (Source AOS) in a 9:1:1.1 ratio. AOSare produced from pectin (Südzucker AG, Mannheim, Germany), with a DP of1-20. It consists of approximately 75% galacturonic acid oligomers,based on total weight;

2) 1 wt % lactoneotetraose (LNnT),

3) 1 wt % 3′-sialyl lactose (3′-SL), or

4) 1 wt % 2′-fucosyllactose (2′-FL).

All groups were compared to the unsupplemented control diet. Dietarysupplementation started 14 days before the first vaccination and lasteduntil the end of the experiment, 31 days after the first vaccination.

Vaccination experiments were performed using Influvac (SolvayPharmaceuticals, Weesp, the Netherlands) from season 2005/2006. The micereceived a primary vaccination and a booster vaccination, consisting ofa subcutaneous (sc) injection of a 1:1 mix of vaccine and adjuvant in atotal volume of 100 μl. The booster vaccination was given at 21 daysafter the primary vaccination. The experiments ended 10 days afterbooster vaccination. Blood samples were taken at the end of theexperiment. Negative control groups that were included receivedinjections with a 1:1 mix of PBS and adjuvant in a total volume of 100μl. To determine the percentage of NK cells, cells were labelled withFITC-labelled anti-mouse CD3 mAb in combination with PE-labelledanti-mouse NK1.1 mAb. NK cell cytotoxicity in spleen cell suspensionswas assayed using standard ⁵¹Cr release assays. Briefly, NK cellcytotoxicity was tested using YAC-1 target cells. The percentage ofspecific ⁵¹Cr release was calculated as the percentage of specificlysis=(experimental release−spontaneous release)/(total detergentrelease−spontaneous release)×100. The spontaneous release values werealways <15% of total lysis.

The percentage of regulatory T cells (Treg) was determined by flowcytometry (FACSCalibur) using allophycocyanin (APC-)labelled anti-mouseCD3 mAb, Pe-Cy5-labeled anti-mouse CD4 mAb and phycoerythrin(PE)-labelled anti-mouse CD25 mAb in combination with intracellularstaining of fluorescein-isotiocyanate (FITC)-labelled Foxp3 mAb,according to the instructions offered by the manufactures (eBiosciences,San Diego, Calif.).

Statistical analysis was performed using GraphPadPrism software.Statistical differences between test and control groups were analysed byANOVA and post hoc Dunnett's test if multiple groups were compared to asingle (control) group. P-values<0.05 were considered significant in allexperiments.

Results

The immunomodulatory effect of three chemically synthesised humanoligosaccharides was compared with GOS/FOS/AOS. Supplementation withGOS/FOS/AOS, LNnT, 2′-FL or 3′-SL resulted in a significant increase ofthe DTH response, a TH1-dependent parameter, compared with control-fedanimals.

Interestingly, the percentage of NK cells in the spleen wassignificantly increased in mice supplemented with human oligosaccharidescompared to control and GOS/FOS/AOS-supplemented mice, see table 1. Thiseffect was highest with 2′-FL. To examine whether the increase in thepercentage of NK cells in human oligosaccharides-supplemented groupsalso correlated with functional activity, NK cell activity was measuredin mouse splenocytes. A significant increased NK cell activity wasdetected in the splenocytes of mice that were supplemented with 2′-FLcompared to controls, see Table 1.

TABLE 1 Effect of dietary NDO similar to human milk NDO on NK cells andNK cell activity. NK cell activity, Dietary % NK 1.1 + % lysis At E:Tintervention cells (SE) ratio 1:50 Sham control 2.5 (0.2) 1.57 Control2.2 (0.1) 1.13 GOS/FOS/AOS 2.5 (0.1) 1.95 LNnT 2.8 (0.1)* 2.14 3′-SL 3.0(0.2)* 1.93 2′-FL 3.7 (0.1)** 2.48* *indicates ρ < 0.05 compared tocontrol group **indicates ρ < 0.01 compared to control group

Finally, the amount of regulatory T cells (Treg) was decreased to thehighest extent with the diet with FL as can be seen in Table 2.

TABLE 2 Effect of dietary NDO similar to human milk NDO on percentage ofregulatory T cells Treg. Dietary Treg percentage intervention (s.e.m.)Sham control 3.6 (0.4) Control 3.9 (0.2) GOS/FOS/AOS 4.1 (0.3) LNnT 3.2(0.2) 3′-SL 2.8 (0.2)* 2′-FL 2.8 (0.2)* *indicates ρ < 0.05 compared tocontrol group

The decrease in regulatory T cells is indicative for a decreasedinhibition of immune response and hence enables an increased vaccinationresponse to the viral antigens. A temporarily decrease of regulatory Tcells can be especially beneficial when vaccination is to occur.

Overall, these results support that oral supplementation with 2′-FLstimulates growth and/or activity of NK cells. These results areindicative for an effect of dietary 2′-FL for enhancing vaccinationresponse, in particular vaccination with viral antigens. These resultsare indicative for an effect of dietary 2′-FL for treating and/orpreventing viral infections.

Example 2

Infant formula for stimulating NK cell activity comprising per 100 ml(13.9 dry weight):

1.4 g protein (whey and casein)

7.3 g digestible carbohydrates (including lactose)

3.6 g fat (vegetable fat, fish oil)

0.8 g non-digestible oligosaccharides of which 80 mg 2′-fucosyllactoseand 640 mg beta-galacto-oligosaccharides, and 80 mgfructo-oligosaccharides

Further are included: choline, myo-inositol, taurine, minerals, traceelements, and vitamins as known in the art.

Example 3

A preferred composition that can be used for the stimulation of naturalkiller cell activity in HIV patients may comprise per 100 g dry weight.

Dietary fibre  5-50 g Fructo-oligosaccharide  5% of total dietary fibreGalacto-oligosaccharide 40% of total dietary fibre Pectin hydrolysate50% of total dietary fibre 2′-FL  5% of total dietary fibre N-acetylcysteine 0.5-5 g Carbohydrate (not dietary fibre)  2-20 g Fat  4-20 g

1-11. (canceled)
 12. A method of stimulating natural killer (NK) cellactivity and/or NK cell proliferation, comprising administering to asubject in need thereof an enteral composition comprisingfucosyllactose, wherein the composition is not human milk.
 13. Themethod according to claim 12, wherein subject is an infant HIV patient,elderly and/or oncology patient.
 14. The method according to claim 12,wherein the subject is an infant.
 15. The method according to claim 12,wherein the composition additionally comprises at least one ofbeta-galacto-oligosaccharides, fructo-oligosaccharides and uronic acidoligosaccharides.
 16. The method according to claim 12, wherein thefucosyllactose is 2′-fucosyllactose.
 17. The method according to claim12, wherein the composition comprises 0.07 to 1 wt % fucosyllactosebased on dry weight of the composition.
 18. The method according toclaim 12, wherein the composition comprises 5 to 50% protein, 15 to 85%digestible carbohydrates and 5 to 50% fat based on total energy.
 19. Amethod of treating and/or preventing viral infections, comprisingadministering to a subject in need thereof an enteral compositioncomprising fucosyllactose, wherein the composition is not human milk.20. The method according to claim 19, wherein subject is an infant HIVpatient, elderly and/or oncology patient.
 21. The method according toclaim 19, wherein the subject is an infant.
 22. The method according toclaim 19, wherein the composition additionally comprises at least one ofbeta-galacto-oligosaccharides, fructo-oligosaccharides and uronic acidoligosaccharides.
 23. The method according to claim 19, wherein thefucosyllactose is 2′-fucosyllactose.
 24. The method according to claim19, wherein the composition comprises 0.07 to 1 wt % fucosyllactosebased on dry weight of the composition.
 25. The method according toclaim 19, wherein the composition comprises 5 to 50% protein, 15 to 85%digestible carbohydrates and 5 to 50% fat based on total energy.
 26. Amethod of enhancing a vaccination response, comprising administering toa subject in need thereof a composition comprising fucosyllactose. 27.The method according to claim 26, wherein subject is an infant HIVpatient, elderly and/or oncology patient.
 28. The method according toclaim 26, wherein the composition additionally comprises at least one ofbeta-galacto-oligosaccharides, fructo-oligosaccharides and uronic acidoligosaccharides.
 29. The method according to claim 26, wherein thefucosyllactose is 2′-fucosyllactose.
 30. The method according to claim26, wherein the composition comprises 0.07 to 1 wt % fucosyllactosebased on dry weight of the composition.
 31. The method according toclaim 26, wherein the composition comprises 5 to 50% protein, 15 to 85%digestible carbohydrates and 5 to 50% fat based on total energy.